DESCRIPTION: (adapted from applicant?s abstract) The investigators propose to use de novo protein synthesis to combine the advantages of organic chemistry and synthetic construction with molecular biology and site directed mutagenesis. Synthetic four alpha helix bundle proteins have been designed and made with extraordinary stability, unique structures, and demonstrated low dielectric interiors. The investigators plan to use the flexibility of simplified, tailored proteins to determine the guidelines for how natural proteins may engineer the burial of charges in their interior, design partial compensation of charges, and direct remaining electric field forces toward functional purpose. They plan to systematically explore the placement of internally placed single, pairs, and other constellations of charge arrangements in the interior structure and determine how dipolar properties of nearby placed residues respond to the charges and in return control them. In addition to the common practice of measuring the response to electric fields from apparent pK shifts of ionizable acid-base amino acid sidechains, they also plan to take advantage of the multiple, perturbing and analytical roles of redox cofactors introduced into the interior of the bundles. Redox cofactors not only can be used to introduce charge in situ by oxidation-reduction, their dual ionizable character of oxidation-reduction (apparent shifts in Em values) and coupled acid-base transitions (pK shifts) provides critical and informative thermodynamic probes of the electric environment of the protein interior. They also bring an independent view of the events through their well-characterized spectroscopic properties. The investigators plan to take the spectroscopic advantage further with explorations to determine the feasibility of measuring electric field strength directly from electrochromic (Stark) shifts on spectra of introduced chromophores such as carotenoid and retinal polyenes and conjugated rings of porphyrins and chlorins. The understanding that will grow out of the work is basic to any description of the role of fields in establishing structure, stability and catalytic function in natural proteins. The use of redox cofactors brings significance to their involvement in signaling, gene regulation, energy conversion and oxidoreductase enzymes, and to the long-standing question of how the properties of a single cofactor can be controlled by different proteins over very large ranges. Such understanding is fundamental to any long-term hope of creating novel proteins that actively control internal electric fields and manipulate chemistry at specific internal sites.